In this proposal we wish to assess as to how far human stem cells suffer from random accumulating changes in the genome, epigenome and transcriptome, measured by comparative, integrated analysis of single cells. This question is relevant not only for aging-related functional decline of organs and tissues, but especially for the validity of using human embryonic or adult stem cells in regenerative medicine. While there is ample evidence for genomic and epigenomic changes in cell populations, including embryonic stem cells during sub-culture, the functional impact of such changes is unclear. This is due to a virtual lack of methods to study cells not as averages across entire populations, but at the level of individual cells, in assays that integrate changes in the transcriptome with genomic and epigenomic changes. Here we propose to use integrative assays for assessing alterations in the genome, epigenome and transcriptome in the same individual cells. This approach requires technology sensitive and precise enough to manipulate individual cells and interrogate them sequentially for various experimental end points. The main question we wish to address here is whether human embryonic stem (hES) cells are capable of withstanding the genetic and epigenetic erosion that occurs in normal, differentiated somatic cells. Since it is hoped that hES cells can be used to replenish organs and tissues, it is extremely important to know whether such cells experience genetic drift and if so the rate and magnitude of spontaneous alterations. Such an approach has never been attempted before and lends itself well to the EUREKA mechanism because it addresses an extremely difficult problem, is unsuitable for regular NIH support due to the high risk involved and the relative lack of preliminary data, is highly innovative with ample use of novel technologies and can be completed within a 4-year time frame. Public Health Relevance: Aging is marked by a degenerative decline in multiple organ systems. Among the promising therapies for this degeneration is the use of stem cells - both adult and embryonic - to repair and regenerate deteriorated tissues and organs. The promise of this approach, however, depends critically on understanding whether stem cells resist or succumb to (epi)genomic damage. Here, we use '(epi)genomic'to indicate either the DNA sequence or chromatin state. Our proposed experiments will generate unique and robust methods for determining how individual cells, stem and differentiated, handle damage to their (epi)genome. They have the potential to shift our understanding of human disease from current views which do not account for individual cell-to-cell variation to a more accurate and useful view of tissue failure even in the absence of strong all- pervasive (epi)genetic changes, which does not occur in the vast majority of humans that experience age- related disease.